Spider silk materials genetically engineered with enzyme activity

Abstract

Event Abstract Back to Event Spider silk materials genetically engineered with enzyme activity Ronnie Jansson1, Christophe M. Courtin2, Martin Östberg3, Mats Sandgren4 and My Hedhammar1, 3 1 Swedish University of Agricultural Sciences, Department of Anatomy, Physiology and Biochemistry, Sweden 2 KU Leuven, Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre, Belgium 3 KTH Royal Institute of Technology, School of Biotechnology, Division of Protein Technology, Sweden 4 Swedish University of Agricultural Sciences, Department of Chemistry and Biotechnology, Sweden Introduction: A common way to accomplish catalytically functional surfaces is to use immobilized enzymes. By covalent attachment of enzymes to a surface, a stable bond between the support and enzyme is created, making the enzyme-surface suitable for repeated and continuous use. The recombinant spider silk protein 4RepCT has previously been shown to self-assemble into silk-like fibers in near-physiological conditions[1]. Moreover, genetic linkage has been used to functionalize 4RepCT with cell-binding[2],[3] and affinity[4] properties. Herein, we have broadened the complexity of silk-linked domains by exploring the concept of enzyme-silk. In the present study, the enzyme xylanase[5] has been genetically fused to 4RepCT, and the silk-like materials produced thereof investigated in terms of enzymatic activity. Materials and Methods: A gene for the enzyme xylanase was fused by genetic linkage to the 4RepCT silk gene, prior to production of soluble xylanase-4RepCT fusion protein in Escherichia coli. After purification using chromatography, the enzyme-silk fusion protein was allowed to form silk-like fibers. Surface coating and foam were also prepared from xylanase-4RepCT. Enzymatic activity of the xylanase-silk materials was determined using a colorimetric reducing-sugar assay (Megazyme, Bray, Ireland). Catalytic activity was also measured after storage at different conditions, as well as after incubation with 70% ethanol and after three consecutive uses (reuse) of the xylanase-silk materials. To investigate the activity in a continuous substrate flow, xylanase-silk fibers were incubated with circulating substrate, after which the activity was determined as the presence of accumulated product at various number of cycles. Results and Discussion: The enzyme-silk protein xylanase-4RepCT could be produced and retrieved after purification as soluble fusion proteins under near-physiological conditions. Xylanase-silk proteins could also form silk-like fibers (Figure 1a), despite the relatively large size of the linked xylanase (20 kD) compared to 4RepCT (23 kDa). An enzyme assay revealed that xylanase in xylanase-silk fibers was catalytically active, although covalently linked to silk[6]. The versatility of xylanase-silk materials was shown by producing 2D surface coating and 3D foam (Figure 1a) that also displayed enzymatic activity. Moreover, the xylanase-silk materials were catalytically active after treatments such as storage, ethanol cleaning and reuse, important stability parameters for functional materials. Xylanase-silk fibers were further demonstrated to be active in a continuous substrate flow, opening for continuous enzyme processing using enzyme-silk fibers (Figure 1b), and not only batch processing. Encouraged by these results, other enzyme-silk materials have been produced, and will also be explored in the context of catalytic activity. By utilizing simultaneous production of the enzyme and the material (silk) as a fusion protein, where each enzyme molecule will be linked to a silk molecule, a stable functional material with a dense enzyme immobilization is achieved. Figure 1. (a) Light microscopy images showing the macroscopic appearance of xylanase-silk fiber (left) and foam (right). Scale bars indicate 1 mm. (b) Absorbance of accumulated product at different substrate cycles from xylanase-silk fibers in a continuous substrate flow. Conclusion: Herein we show a proof-of-principle for the concept of enzyme-silk, in which the recombinant silk module 4RepCT has been fused to an enzyme by genetic linkage. From such enzyme-silk fusion proteins, 2D and 3D silk materials with a dense and stable immobilization of catalytically active enzymes can be assembled. We therefore envision enzyme-silk materials to find its applicability in areas as multienzyme reaction systems, as well as biosensors where the dense enzyme immobilization can increase the sensitivity. Soluble 4RepCT silk protein was kindly provided by Spiber Technologies AB. The Swedish Research Council, FORMAS and Knut & Alice Wallenberg Foundation are acknowledged for financial support.